4. Sample preparation
Sample (5.0 g) was weighed into a 250-mL glass tube and 20〜25 mL of water and 100 μL of internal standard solution (100 μg/mL AA-1-13C solution) were added to the sample. The mixture was allowed to stand for 30 min with occasional shaking. Acetone (50 mL) was added and the mixture was homogenized for 1 min with a homogenizer. After filtrating the homogenate through a glass filter (GA-100), the residue on the filter was washed with 30 mL of acetone. The filtrate was concentrated to less than 15 mL by a concentrator at 35℃. The extract was transferred in a 50-mL centrifugal tube, washed twice with each 20 mL of dichloromethane by shaking for 1 min and centrifuged at 2,700 rpm for 5 min. The extract was made up to 15 mL with water.
An aliquot of the extract (3 mL, equivalent to 1.0 g of sample) was passed through a C18 cartridge column with following washing with 2 mL of water. All the eluate was collected and made up to 5 mL with water. An aliquot of the eluate (2 mL, equivalent to 0.4 g of sample) was passed through an ACCUCAT column connected to PSA column with following washing with 2 mL of water. All the eluate was collected and made up to 4 mL with water to prepare the test solution.

5. Determination and confirmation of AA
AA was determined by LC/MS(SIR). The quantification was performed by the comparison of the peak area ratios of m/z 72 (AA) and m/z 73 (AA-1-13C) for the test solution with those for the calibration standard. The peak area ratio of m/z 55 (AA) and m/z 56 (AA-1-13C) was used for the qualification. The recovery of internal standard was calculated from the peak areas of AA-1-13C in the test solution and the calibration standard.
The calibration standard (AA=0, AA-1-13C=200 ng/mL) contained a small quantity of AA as an impurity of AA-1-13C. Therefore, the limit of detection (LOD) was defined as 3 times the SD of the AA peak area of calibration standard (AA=0, AA-1-13C=200 ng/mL). Similarly, the limit of quantification (LOQ) was defined as 10 times the SD.